Maryrose J. Conklyn

ATP Acts as an Agonist to Promote Stimulus-Induced Secretion of IL-1β and IL-18 in Human Blood

Cultured monocytes and macrophages stimulated with LPS produce large quantities of proIL-1β, but release little mature cytokine to the medium. The efficiency at which the procytokine is converted to its active 17-kDa species and released extracellularly is enhanced by treating cytokine-producing cells with a secretion stimulus such as ATP or nigericin. To determine whether this need for a secretion stimulus extends to blood, individual donors were bled twice daily for 4 consecutive days, and the collected blood samples were subjected to a two-step IL-1 production assay. LPS-activated blood samples generated cell-free IL-1β, but levels of the extracellular cytokine were greatly increased by subsequent treatment with ATP or nigericin. Specificity and concentration requirements of the nucleotide triphosphate effect suggests a P2X7 receptor involvement. Quantities of IL-1β generated by an individual donor’s blood in response to the LPS-only and LPS/ATP stimuli were relatively consistent over the 4-day period. Between donors, consistent differences in cytokine production capacity were observed. Blood samples treated with ATP also demonstrated enhanced IL-18 production, but TNF-α levels decreased. Among leukocytes, monocytes appeared to be the most affected cellular targets of the ATP stimulus. These studies indicate that an exogenous stimulus is required by blood for the efficient production of IL-1β and IL-18, and suggest that circulating blood monocytes constitutively express a P2X7-like receptor.

The Novel JAK-3 Inhibitor CP-690550 Is a Potent Immunosuppressive Agent in Various Murine Models

JAK‐3 has been shown to play a key role in cytokine signaling via γc, e.g. IL‐2, 4, 7, 9, 15, 21. The current study describes the immunosuppressive effects of CP‐690550, a novel, small molecule inhibitor of JAK‐3, in various murine models. In vitro, CP‐690550 effectively inhibited a murine mixed lymphocyte reaction (MLR) (IC50= 91 nm). Mice chronically dosed with CP‐690550 (1.5–15 mg/kg/day) demonstrated dose‐ and time‐dependent alterations in lymphocyte subsets when examined by flow cytometry. The most dramatic change observed was a 96% reduction in splenic NK1.1 + TCRβ– cell numbers following 21 days of treatment. Delayed‐type hypersensitivity (DTH) responses in sensitized mice were reduced in a dose‐dependent manner following treatment with the JAK‐3 inhibitor (1.87–30 mg/kg, s.c.). Extended survival of neonatal Balb/c hearts implanted into the ear pinna of MHC mismatched C3H/HEN mice was observed with CP‐690550 monotherapy (10–30 mg/kg/day), but improved upon combination with cyclosporin (10 mg/kg/day). These data support the participation of JAK‐3 in various lymphocyte homeostatic functions in mature mice. Furthermore, the ability of CP‐690550 to extend cardiac allograft survival in murine models suggests it may afford a new treatment for prevention of transplant rejection.

The JAK-3 inhibitor CP-690550 is a potent anti-inflammatory agent in a murine model of pulmonary eosinophilia.

Janus kinase 3 (JAK-3) is a tyrosine kinase that has been shown to participate in the signaling of several cytokines that are believed to play a role in allergic airway disease, e.g. IL-2, 4 and 9. The current study describes the immunosuppressive effects of CP-690550, a novel, small molecule inhibitor of JAK-3, in a murine model of allergic pulmonary inflammation. In vitro, CP-690550 potently inhibited IL-4 induced upregulation of CD23 (IC(50)=57 nM) and class II major histocompatibility complex (MHCII) expression (IC(50)=71 nM) on murine B cells. Repeat aerosol exposure to ovalbumin in wild-type mice sensitized to the antigen resulted in preferential recruitment of Th2-like cells (IL-4+ and IL-5+) into bronchoalveolar lavage fluid (BAL). The importance of IL-4 in the development of pulmonary eosinophilia was supported by a marked (90%) reduction in the influx of these cells in IL-4KO mice similarly sensitized and ovalbumin exposed. Animals dosed with CP-690550 (15 mg/kg/d) during the period of antigen sensitization and boost demonstrated marked reductions in BAL eosinophils and levels of IL-13 and eotaxin following ovalbumin aerosol exposure. The JAK-3 inhibitor (1.5-15 mg/kg/d) also effectively reduced the same parameters when administered during the period of antigen challenge. In contrast, the calcineurin inhibitor tacrolimus (10 mg/kg) was effective only when administered during the period of ovalbumin aerosol exposure. These data support the participation of JAK-3 in processes that contribute to pulmonary eosinophilia in the allergic mouse model. CP-690550 represents an intriguing novel therapy for treatment of allergic conditions associated with airway eosinophilia including asthma and rhinitis.

The JAK3 inhibitor CP-690550 selectively reduces NK and CD8+ cell numbers in cynomolgus monkey blood following chronic oral dosing

Janus kinase 3 (JAK3) is a cytoplasmic tyrosine kinase associated with the common γ chain, an integral component of cytokine receptors of the interleukin (IL)‐2 family, including IL‐4, ‐7, ‐9, ‐15, and ‐21. CP‐690550 is a JAK3 inhibitor with immunosuppressive properties under development for transplantation. We evaluated alterations in circulating lymphocyte subsets in cynomolgus monkey blood following chronic (3‐week), oral CP‐690550 administration. Natural killer (NK) and CD8+ T cell numbers were reduced in a dose‐ and time‐dependent manner; the latter was a primary effect on memory subsets. CD4+ T and B cell numbers were unaffected or slightly increased, respectively. NK cell numbers were reduced ∼80% (vs. 35% in vehicle‐treated animals) and returned to baseline levels within 3 weeks following treatment cessation. CD8+ T cells declined by a maximum 43% (vs. 25% for vehicle‐treated animals) but rebounded significantly (300%) within 2 weeks after the last dose. Although CP‐690550 did not result in reduction of CD4+ T cell number, these cells also increased (225%) within 2 weeks of treatment cessation. IL‐15 is important for maintaining homeostasis of these cell types, and CP‐690550 inhibited IL‐15‐induced CD69 expression in NK cells [inhibitory concentration 50% (IC50)=48.0±8.4 nM] and CD8+ T cells (IC50=16.2±1.5 nM).

CP-481,715, a Potent and Selective CCR1 Antagonist with Potential Therapeutic Implications for Inflammatory Diseases

The chemokines CCL3 and CCL5, as well as their shared receptor CCR1, are believed to play a role in the pathogenesis of several inflammatory diseases including rheumatoid arthritis, multiple sclerosis, and transplant rejection. In this study we describe the pharmacological properties of a novel small molecular weight CCR1 antagonist, CP-481,715 (quinoxaline-2-carboxylic acid [4(R)-carbamoyl-1(S)-(3-fluorobenzyl)-2(S),7-dihydroxy-7-methyloctyl]amide). Radiolabeled binding studies indicate that CP-481,715 binds to human CCR1 with a Kd of 9.2 nm and displaces 125I-labeled CCL3 from CCR1-transfected cells with an IC50 of 74 nm. CP-481,715 lacks intrinsic agonist activity but fully blocks the ability of CCL3 and CCL5 to stimulate receptor signaling (guanosine 5′-O-(thiotriphosphate) incorporation; IC50 = 210 nm), calcium mobilization (IC50 = 71 nm), monocyte chemotaxis (IC50 = 55 nm), and matrix metalloproteinase 9 release (IC50 = 54 nm). CP-481,715 retains activity in human whole blood, inhibiting CCL3-induced CD11b up-regulation and actin polymerization (IC50 = 165 and 57 nm, respectively) on monocytes. Furthermore, it behaves as a competitive and reversible antagonist. CP-481,715 is >100-fold selective for CCR1 as compared with a panel of G-protein-coupled receptors including related chemokine receptors. Evidence for its potential use in human disease is suggested by its ability to inhibit 90% of the monocyte chemotactic activity present in 11/15 rheumatoid arthritis synovial fluid samples. These data illustrate that CP-481,715 is a potent and selective antagonist for CCR1 with therapeutic potential for rheumatoid arthritis and other inflammatory diseases.

Tyrosine phosphorylation is an early signaling event common to Fc receptor crosslinking in human neutrophils and rat basophilic leukemia cells (RBL-2H3)

Phosphotyrosine-containing proteins were detected by western blotting of whole cell lysates of purified human neutrophils or rat basophilic leukemia cells (RBL-2H3) using a polyclonal anti-phosphotyrosine antibody. When either cell type was stimulated with the appropriate Fc crosslinking agent, heat-aggregated IgG for the neutrophil or DNP-HSA for the IgE-sensitized RBL-2H3, a rapid increase in the phosphotyrosine content of several proteins was observed. The kinetics and specificity of both responses suggest that Fc receptor crosslinking activates a receptor-associated tyrosine kinase, probably a member of the src family of tyrosine protein kinases. The subsequent tyrosine phosphorylation events are likely to be important in Fc receptor-mediated stimulus-response coupling in inflammatory cells.

Characterization of the pharmacological profile of the potent LTB4 antagonist CP-105,696 on murine LTB4 receptors in vitro.

1 Binding of [3H]‐leukotriene B4 ([3H]‐LTB4) to murine spleen membranes (MSM) was determined. 2 Scatchard analyses of [3H]‐LTB4 binding indicated the presence of high (KD1 = 1.7 nM) and low (KD2 = 7.5 nM) affinity receptors on MSM with Bmax. values of 151 fmol mg−1 protein (Bmax1) and 354 fmol mg−1 protein (Bmax2), respectively. 3 CP‐105,696, a potent LTB4 antagonist, inhibited [3H]‐LTB4 (0.67 nM) binding to the high affinity receptor on MSM, IC50 = 30.2 nM, Ki = 17.7 nM with a Hill coefficient of 0.93. 4 Scatchard analyses of [3H]‐LTB4 binding to MSM in the presence of CP‐105,696 indicated that the high‐affinity receptor was inhibited in a non‐competitive manner and the low‐affinity receptor in a competitive manner. 5 Isolated peripheral blood murine neutrophils (MN) responded chemotactically to LTB4, EC50 = 2.5 nM. CP‐105,696 blocked this response, IC50 = 2.3 nM. When examined over a full concentration‐response range of LTB4, CP‐105,696 inhibited chemotaxis in a non‐competitive manner. 6 Murine neutrophils in anticoagulated whole blood upregulated the integrin, complement receptor type 3 (CD11b/CD 18, Mac‐1) in response to LTB4, EC50 = 20 nM and this was inhibited by CP‐105,696 in a competitive manner. 7 These results provide evidence that MSM have specific binding sites for LTB4, and as exemplified by CP‐105,696, that these receptors may be useful for determining the potency and nature of antagonism of novel LTB4 receptor antagonists.

Functional Effects of Eotaxin Are Selectively Upregulated on IL-5 Transgenic Mouse Eosinophils

Synergistic interactions between cytokines, chemokines and adhesion molecules may facilitate the selective recruitment of eosinophils into sites of allergic inflammation. Ovalbumin-sensitized IL5TG mice responded to antigen challenge with robust airway eosinophilia 24 and 72 hr post-exposure. Adhesion molecule expression and functional responsiveness of immune cells derived from IL5TG mice to various inflammatory mediators were evaluated. IL5TG-derived eosinophils, but not neutrophils, expressed higher levels of CD49d and CD11b relative to WT. Functional responsiveness to eotaxin was increased in IL5TG eosinophils as demonstrated by a 10× increase in its potency in producing actin polymerization and 3× increase in CD11b upregulation relative to WT. These data are consistent with increased CCR3 expression on IL5TG eosinophils. Responsiveness of eosinophils to LTB4 or MIP-1α was similar between WT and IL-5TG mice. These data provide evidence of synergy between eosinophil-specific cytokines and chemokines that may promote accumulation of this cell type under conditions of allergic inflammation in vivo.

Inhibition of IgE-mediated N-acetylglucosaminidase and serotonin release from rat basophilic leukemia cells (RBL-2H3) by tenidap : a novel anti-inflammatory agent

Tenidap [(Z)-5-chloro-2,3-dihydro-3-(hydroxy-2-thienylmethylene)-2-oxo-1H- indole-1-carboxamide] is a novel anti-inflammatory compound of the oxindole class that currently is undergoing clinical evaluation in man. Here we demonstrate that tenidap inhibits (IC50 = approximately 10 microM) IgE-mediated secretion of granule constituents from the rat mast cell tumor line RBL-2H3. The inhibitory effect is rapid in onset, readily reversible, and appears to be unique when compared to a representative selection of other acidic (carboxylic acids, pyrazoles and oxicams) nonsteroidal anti-inflammatory compounds.

Optimization and validation of a flow cytometric method for immunophenotyping peripheral blood lymphocytes from cynomolgus monkeys (Macaca fascicularis)

BACKGROUND Guidelines published by the Food and Drug Administration and Center for Human Medicinal Products describe the need to assess immunotoxic effects in nonclinical studies that evaluate drug toxicity, including the use of immunophenotyping to measure immunotoxicity. We are not aware of previous studies, however, that have validated methods for immunophenotyping peripheral blood lymphocyte subsets in whole blood samples from cynomolgus monkeys. OBJECTIVE The purpose of this study was to optimize and validate a flow cytometric assay for immunophenotyping lymphocytes in the peripheral blood of cynomolgus monkeys. METHODS A series of prevalidation experiments were done to determine optimal reagents, volumes, timing, and other procedural details of the flow cytometric assay. Using the optimized method, we then determined precision, interindividual variation, laboratory-to-laboratory variability, and sample stability. Stabilized human blood was used as a positive control for staining, processing, and analysis. The percentage and number of pan-T cells (CD3+), T-helper cells (CD3+4+), T cytotoxic/suppressor cells (CD3+8+), natural killer cells (CD3-16+), and B-cells (CD3-20+) were determined in 146 male and 140 female, clinically healthy monkeys and reference intervals were calculated. RESULTS By doing 4-color staining with a lyse-wash method, intra- and interassay precision were <5% for all lymphocyte subsets. Variability between technicians and laboratories was minimal (CVs<3%). Samples were stable for up to 24 hours after staining and fixing. CONCLUSIONS The validated method is extremely robust and can be performed under good laboratory practice conditions to support nonclinical studies. Reference intervals for lymphocyte subsets were similar to those previously reported.